Methods of modulation of branched chain acids and uses thereof

ABSTRACT

A method of modulating plasma levels of branched chain amino acids and branched chain alpha-keto acids is disclosed, wherein an ammonia scavenger compound or a salt thereof, for example phenylbutyrate or an even numbered congener thereof or a salt thereof, is administered to an individual in need thereof. In various methods, a decrease in plasma levels of branched chain amino acids and branched chain alpha-keto acids is effected to treat individuals suffering from an inborn error in metabolism of amino acids, such as Maple Syrup Urine Disease, for example.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Non-Provisional applicationSer. No. 15/653,155 filed Jul. 18, 2017, which is a continuation of U.S.Non-Provisional application Ser. No. 14/745,205 filed on Jun. 19, 2015,which is a continuation of U.S. Non-Provisional application Ser. No.13/386,549 filed May 7, 2012, which is a national phase applicationunder 35 U.S.C. § 371 that claims the benefit of priority fromInternational Application No. PCT/US2010/043240 filed Jul. 26, 2010,which claims priority to U.S. Provisional Application No. 61/228,485,filed Jul. 24, 2009, all of which are incorporated herein by referencein their entirety.

BACKGROUND OF THE INVENTION

Branched chain amino acids (BCAAs) account for about 40% of theessential amino acids in healthy subjects and must be acquired through awell-balanced diet. Branched-chain amino acids are toxic in excess butare required for protein synthesis. The normal plasma levels in childrenfor isoleucine, leucine, and valine, range from 37 to 40 μmol/L, 70 to170 μmol/L, and 160 to 350 μmol/L, respectively. In adults, the normalplasma levels for isoleucine, leucine, and valine are 42 to 100 μmol/L,66 to 170 μmol/L, and 150 to 310 μmol/L, respectively. The BCAAs haveimportant physiologic functions in addition to their role as proteinprecursors. In peripheral tissues such as skeletal muscle, BCAAs arenitrogen donors for the synthesis of alanine and glutamine, thus movingnitrogen derived from muscle amino acid oxidation to the liver for ureasynthesis. In addition, leucine acts as an anabolic nutrient signalinfluencing both insulin secretion by the β-cells of the pancreas andprotein synthesis in skeletal muscle and some other tissues. In order tobalance the body's need for BCAAs with the supply of BCAAs from thediet, the BCAA catabolic pathway is tightly regulated. The catabolicpathways of BCAAs have two common steps. The first is a reversibledeamination catalyzed by vitamin-B₆-dependent branched-chainaminotransferase (BCATs) to produce the corresponding branched-chainα-keto acid (BCKAs). The second is the irreversible oxidativedecarboxylation of the BCKAs, accomplished in a large part by control ofthe activity of the branched-chain α-keto acid dehydrogenase complex(BCKDC).

Deregulation of branched chain amino acid catabolism leads to an inbornerror of metabolism in newborns known as maple syrup urine disease(MSUD). MSUD, also called branched-chain ketoaciduria, is an autosomalrecessive disorder, typically diagnosed shortly after birth. It iscaused by defects in BCKDC. The defect thus results in an accumulationof the BCAAs, namely, leucine, valine, isoleucine, and their respectiveα-keto acids (α-ketoisocaproate, α-ketoisovalerate, andα-keto-β-methylvalarate) in cells and body fluids. Accumulation of thesethree amino acids and their corresponding keto acids leads toencephalopathy and progressive neurodegeneration in untreatedindividuals.

The present invention addresses a long-felt need for therapies that areeffective in regulating the BCAA catabolism, such as in MSUD, that canbe used independently or in conjunction with dietary compliance.

SUMMARY OF THE INVENTION

In particular embodiments of the invention, there are methods andcompositions for treating an individual (including a mammal, such as ahuman, dog, cat, horse, pig, goat, or sheep, for example) for an inbornerror in amino acid metabolism with one or more ammonia scavengers. Inspecific aspects, the individual has a medical condition as a result ofthe inborn error in amino acid metabolism. Although the invention incertain cases is applicable to an inborn error in any amino acidmetabolism, in specific embodiments the amino acids are branched chainamino acids. In particular cases, the individual has an inborn error inmetabolism of leucine, valine, or isoleucine. In specific embodiments,the individual has MSUD, although in other embodiments the individualhas hypervalinemia, isobutyryl-CoA dehydrogenasedeficiency,⋅beta-ketothiolase deficiency, 2-Methylbutyryl-CoAdehydrogenase deficiency, hypermethioninemia, homocystinuria,cystathioninuria, isovaleric acidemia, 3-Methylcrotonyl-CoA carboxylasedeficiency, or 3-hydroxy-3-methylglutaryl-CoA lyase deficiency. Inspecific embodiments, the individual has Fragile X, tuberous sclerosis,Rett syndrome, autism, or diabetes. In some embodiments, the ammoniascavenger is one or a combination of a composition that contains or ismetabolized to phenylacetate. In particular matters, the ammoniascavenger is phenylbutyrate, BUPHENYL® (sodium phenylbutyrate),AMMONAPS®, butyroyloxymethyl-4-phenylbutyrate, glyceryltri-[4-phenylbutyrate] (HPN-100), esters, ethers, and acceptable salts,acids and derivatives thereof, for example. In specific embodiments ofthe invention, the composition for treatment has ammonia scavengingactivity and/or has histone deacetylase inhibitor activity.

In some embodiments of the invention, there is a method of decreasingplasma levels of at least one of a branched chain amino acid or branchedchain alpha-ketoacid comprising administering to an individual in needthereof a therapeutically effective amount of an ammonia scavengercompound or a pharmaceutically acceptable salt thereof, where the amountof the compound administered stimulates the baseline enzymatic activityof the branched chain dehydrogenase enzyme complex protein to levelseffective in achieving target plasma levels of the branched chain aminoacid or branched chain alpha-ketoacid for the individual.

Also described herein, in specific aspects, is a method of decreasingplasma levels of at least one of a branched chain amino acid or abranched chain alpha-ketoacid comprising administering to an individualin need thereof a therapeutically effective amount of at least onecompound of the formula:

wherein n is 0, 2, 4, 6 or 8, or

a pharmaceutically acceptable salt or ester or prodrug thereof, andwhere the amount of the compound administered stimulates the baselineenzymatic activity of the branched chain dehydrogenase enzyme complexprotein to levels effective in achieving target plasma levels of thebranched chain amino acid and/or branched chain alpha-ketoacid for theindividual. In an alternative embodiment, the levels of BCAA and/or BCKAare not targeted by methods of the invention.

Additionally disclosed herein, in certain embodiments, is a method forselecting, identifying or screening for a compound useful for decreasingblood plasma levels of branched chain amino acids or branched chainalpha-ketoacid, comprising the selection or identification of a compoundcapable of decreasing the phosphorylation of the E1α subunit of branchedchain dehydrogenase enzyme protein complex at position Ser293 andSer303; wherein the decrease in phosphorylation of the subunits at thepositions is effective in increasing the enzymatic activity of theenzyme from baseline activity, in particular embodiments of theinvention.

Additionally, provided in specific cases is a method of treatment forMSUD comprising administering to an individual in need thereof atherapeutically effective amount of a compound or a pharmaceuticallyacceptable salt or ester or prodrug thereof, wherein the amount of thecompound administered is effective in dephosphorylation of the S293 andS303 residues of the E1α subunit of branched chain dehydrogenase enzymecomplex protein, and the decrease in phosphorylation of the subunits atthe positions is effective in increasing the enzymatic activity of theenzyme from baseline activity in the individual.

In one embodiment of the invention, there is a method of treating anindividual for an inborn error of metabolism, comprising the step ofadministering an ammonia scavenger to the individual. In a specificembodiment, the individual has maple syrup urine disease (MSUD). In anadditional specific embodiment, the ammonia scavenger compound isphenylbutyrate or a salt or ester or prodrug thereof. In some cases, themethod further comprises restricting the dietary branched chain aminoacid intake in said individual.

In another embodiment of the invention, there is a method of decreasingplasma levels of at least one of a branched chain amino acid or branchedchain alpha-ketoacid comprising administering to an individual in needthereof a therapeutically effective amount of an ammonia scavengercompound or a pharmaceutically acceptable salt thereof; and assaying fora decrease in plasma levels of at least one of said branched chain aminoacid or branched chain alpha-ketoacid. In specific aspects, the amountof the compound administered stimulates the baseline enzymatic activityof the branched chain dehydrogenase enzyme complex protein to levelseffective in achieving target plasma levels of said branched chain aminoacid or branched chain alpha-ketoacid for said individual. In certaincases, both branched chain amino acid and branched chain alpha-ketoacidplasma levels are decreased.

The branched chain dehydrogenase enzyme complex protein enzymaticactivity is increased by at least about 5% over the baseline enzymaticactivity in the individual, in particular embodiments of the invention.The individual in need thereof is an individual with an inborn error ofmetabolism, and in certain cases the inborn error of metabolism is MSUD,such as the types selected from the classical form, the intermediateform, the intermittent form and the thiamine-responsive form of thedisease.

In specific aspects of the invention, the branched chain amino acid isat least one of leucine, isoleucine and valine. In certain cases, thebranched chain alpha-ketoacid is at least one of keto-isocaproic acid,keto-methylvaleric acid, and ketoisovaleric acid. In some aspects, theammonia scavenger compound is phenylbutyrate or a salt or ester orprodrug thereof. The salt is the sodium salt, calcium salt, lithium saltor a potassium salt, in certain cases.

In particular embodiments of the invention, phenylbutyrate or salt orester or prodrug thereof causes decreased phosphorylation of the S293and S303 residues of the E1 subunit of the branched chain dehydrogenaseenzyme complex protein.

In one embodiment of the invention, there is a method of decreasingplasma levels of at least one of a branched chain amino acid or branchedchain alpha-ketoacid comprising administering to an individual, in needthereof, a therapeutically effective amount of at least one compound ofa particular formula or a pharmaceutically acceptable salt thereof, andassaying for a decrease in plasma levels of at least one of saidbranched chain amino acid or branched chain alpha-ketoacid. In specificembodiments, the amount of the compound administered stimulates thebaseline enzymatic activity of the branched chain dehydrogenase enzymecomplex protein to levels effective in achieving target plasma levels ofbranched chain amino acid and/or branched chain alpha-ketoacid for saidindividual. In specific embodiments, the compound is phenylbutyrate or asalt or ester or prodrug thereof and in further specific embodiments thephenylbutyrate or salt or ester or prodrug thereof causes decreasedphosphorylation of the S293 and S303 residues of the E1 subunit of thebranched chain dehydrogenase enzyme complex protein. In specificembodiments, the branched chain amino acid is at least one of leucine,isoleucine and valine and the branched chain alpha-ketoacid is at leastone of keto-isocaproic acid, keto-methylvaleric acid, and ketoisovalericacid. The compound is administered orally, intra-peritoneally orintravenously, in particular aspects of the invention.

In some cases, an individual in need thereof is an individual with highplasma levels of the branched chain amino acids as compared to levels ofbranched chain amino acids in a healthy individual. In certain aspects,high plasma levels of branched chain amino acids are due to an inbornerror of metabolism, such as MSUD, which may be selected from a groupconsisting of the classical form, the intermediate form, theintermittent form and the thiamine-responsive form of the disease. Themethod may further comprise restricting the dietary branched chain aminoacid intake in said individual.

In one embodiment of the invention, there is a method for screening fora compound useful for decreasing blood plasma levels of branched chainamino acids or branched chain alpha-ketoacid, comprising selecting oridentifying a compound capable of decreasing the phosphorylation of theE1 subunit of branched chain dehydrogenase enzyme protein complex atposition Ser293 and Ser303; thereby increasing the enzymatic activity ofsaid enzyme from baseline enzymatic activity. In particular embodiments,the method comprises providing the E1 subunit of branched chaindehydrogenase enzyme complex protein or a fragment thereof comprising atleast 50 consecutive amino acids that include phosphorylated Ser293 andSer303 residues or fragment thereof; contacting a candidate compoundwith said protein or fragment thereof; and, selecting the candidatecompound that causes dephosphorylation of Ser293 and Ser303 of the E1subunit of branched chain dehydrogenase enzyme complex. In otherspecific embodiments, the method comprises contacting a candidatecompound with a cell expressing the E1 subunit of branched chaindehydrogenase enzyme complex protein and a branched chain amino aciddehydrogenase kinase; assessing the amount of E1 subunit phosphorylatedat position S293 and S303; and, selecting the candidate compound thatdecreases the phosphorylation of E1 subunit of branched chaindehydrogenase enzyme complex in comparison with a control cell which hasnot been contacted with the candidate compound.

In certain embodiments of the invention, there is a method of treatmentfor MSUD comprising administering to an individual, in need thereof, atherapeutically effective amount of a compound or a pharmaceuticallyacceptable salt thereof, wherein the amount of said compoundadministered is effective in dephosphorylation of the S293 and S303residues of the E1 subunit of branched chain dehydrogenase enzymecomplex protein thereby increasing the enzymatic activity of said enzymefrom its baseline activity in said individual. In a specific embodiment,the method further comprises restricting the dietary branched chainamino acid intake in said individual. The compound is phenylbutyrate ora salt or ester or prodrug thereof, in particular aspects. The salt is asodium salt, a calcium salt, a lithium salt or a potassium salt, incertain cases. The compound is administered orally, intra-peritoneallyor intravenously, in particular embodiments. In certain embodiments, theMSUD is selected from a group consisting of the intermediate form,intermittent form and the thiamine-responsive form of the disease.

The foregoing has outlined some of the features and technical advantagesof the present invention in order that the detailed description of theinvention that follows may be better understood. Additional features andadvantages of the invention will be described hereinafter which form thesubject of the claims of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and aspects of the present inventionwill be best understood with reference to the following detaileddescription of a specific embodiment of the invention, when read inconjunction with the accompanying drawings, wherein:

FIG. 1 is a graph representing levels of branched chain amino acids(BCAAs) in control subjects (N=3) at baseline and then after treatmentwith sodium phenylbutyrate (PBA). Abbreviations: Ile=Isoleucine;Leu=Leucine; Val=Valine. *: p<0.05. Values are average of three timepoints after two days of treatment. Study subjects received PBA at thedose of 10 gram/m²/day divided in four equal doses every six hours forthree days. The subjects received a constant protein intake of 0.6grams/kg/day as a combination of BCAA-free formula and whole protein.Measurements were performed on day three at three different time pointsseparated by 30 minutes in the fed state. Note that y-axis representsconcentrations comparing baseline to three days of treatment. All threesubjects show significantly decreased levels for all three BCAAs(leucine, isoleucine and valine) following PBA treatment.

FIG. 2 is a graph representing levels of branched chain ketoacids(BCKAs) in control subjects (N=3) at baseline and then after treatmentwith sodium phenylbutyrate. Abbreviations: KMV=α-keto-β-methylvalerate;KIC=α-ketoisocaproate; KIV=α-ketoisovalerate. *: p<0.05. Values areaverage of three time points after two days of treatment. Subjects wereall on the same steady state protein intake for three days at testing.Study subjects received PBA at the dose of 10 gram/m²/day divided infour equal doses every six hours for three days. The subjects received aconstant protein intake of 0.6 grams/kg/day as a combination ofBCAA-free formula and whole protein. Measurements were performed on daythree at three different time points separated by 30 minutes in the fedstate. Note that y-axis represents concentrations comparing baseline tothree days of treatment. All three subjects decreased their BCKAs.

FIG. 3 is a graph representing levels of branched chain amino acids(BCAAs) in MSUD patients (N=5) at baseline and then after treatment withsodium phenylbutyrate. Values are average of three time points after twodays of treatment. Patients were all on steady state protein intake forthree days at testing. Study subjects received PBA at the dose of 10gram/m²/day divided in four equal doses every six hours for three days.The subjects received a constant protein intake of 0.6 grams/kg/day as acombination of BCAA-free formula and whole protein. Measurements wereperformed on day three at three different time points separated by 30minutes in the fed state. Abbreviations: Ile=Isoleucine; Leu=Leucine;Val=Valine. *: p<0.05. Note that y-axis represents concentrationscomparing baseline to three days of treatment. Three of five MSUDpatients were “responders” and decreased their BCAAs.

FIG. 4 is a graph representing levels of branched chain ketoacids(BCKAs) in MSUD patients (N=5) at baseline and then after treatment withsodium phenylbutyrate. Values are average of three time points after twodays of treatment. Patients were all on steady state protein intake forthree days at testing. The subjects received a constant protein intakeof 0.6 grams/kg/day as a combination of BCAA-free formula and wholeprotein. Study subjects received PBA at the dose of 10 gram/m²/daydivided in four equal doses every six hours for three days. Measurementswere performed on day three at three different time points separated by30 minutes in the fed state. Abbreviations: KMV=α-keto-β-methylvalerate;KIC=α-ketoisocaproate; KIV=α-ketoisovalerate. *: p<0.05. Note thaty-axis represents concentrations comparing baseline to three days oftreatment. Three of five MSUD patients were “responders” and decreasedtheir BCKAs.

FIG. 5 is a Western blot analysis of liver extract using an antibodyagainst the phosphorylated form of the E1 subunit of BCKDC from C57B6mice (N=5 mice per group) treated with phenylbutyrate or placebo. Toinvestigate the effect and mechanism of phenylbutyrate on the BCKD, mice(n=5) were treated with saline or 50 mg/kg/day of phenylbutyrate dividedinto three administrations for three consecutive days and after threedays of treatment they were sacrificed for analyses. Proteins wereextracted from mouse livers and a western blot performed using ananti-phosphoserine BCKDC antibody, the anti-BCKDC-E1 antibody, and theanti-BCKDC-E2 antibody. Each lane corresponds to the liver extract froman independent mouse (from #1 to #3). The phosphorylated form of theBCKD is significantly reduced in the phenylbutyrate treated mice ascompared to the placebo group.

FIG. 6 shows BCKDC transcript levels, as assessed by quantitativeRT-PCR, isolated from liver and muscle cells of C57B6 mice (N=5 mice pergroup) treated with either placebo or phenylbutyrate. The mice (n=5)were treated with saline or 50 mg/kg/day of phenylbutyrate divided intothree administrations for three consecutive days and after three days oftreatment they were sacrificed for analyses. The effect ofphenylbutyrate is not on RNA expression of the respective BCKDCsubunits.

FIGS. 7A-7C. FIGS. 7A and 7B show leucine oxidation in lymphoblast cellsfrom control subjects (C-660, C-661) and MSUD patients (P-1, -2, -3, -4,-5) untreated or treated with 1 mM phenylbutyrate for 48 h. Leucineoxidation was measured by using radioactive assay as described inMaterials and Methods and is expressed in pmol CO2 released/min/mgprotein. Values are means±SD (n=3), * p≥0.05, **p≥0.01. FIG. 7C showsWestern blotting of BCKD complex (E1α-phospho{P}/E1α and E2 subunits),BCATm and BCATc in lymphoblast cells from control subjects (C-660,C-661) and MSUD patients (P-1, -2, -3, -4, -5) untreated or treated with1 mM phenylbutyrate (PB) for 48 h. β-tubulin is used as an internalcontrol. Images are representative of three independent experiments.

FIGS. 8A-8C. FIGS. 8A and 8B show leucine oxidation in presence of CICin lymphoblast cells from control subjects (C-660, C-661) and MSUDpatients (P-1, -2, -3, -4, -5) untreated or treated with 1 mMphenylbutyrate for 48 h. Leucine oxidation was measured by usingradioactive assay as described in Materials and Methods and is expressedin pmol CO2 released/min/mg protein. CIC was added to all reactions in 1mM concentration. Values are means±SD (n=3), * p≥0.05, **p≥0.01. FIG. 8Cshows Western blotting of BCKD complex (E1α-phospho{P}/E1α subunit) inlymphoblast cells from control subjects (C-660,C-661) and MSUD patients(P-1, -2, -3, -4, -5) untreated or treated with 1 mM CIC orphenylbutyrate (PB) for 2 or 48 h, respectively. Images arerepresentative of two independent experiments.

DETAILED DESCRIPTION OF THE INVENTION

In the following description, certain details are set forth such asspecific quantities, sizes, etc. so as to provide a thoroughunderstanding of the present embodiments disclosed herein. However, itwill be obvious to those skilled in the art that the present disclosuremay be practiced without such specific details. In many cases, detailsconcerning such considerations and the like have been omitted inasmuchas such details are not necessary to obtain a complete understanding ofthe present disclosure and are within the skills of persons of ordinaryskill in the relevant art.

I. DEFINITIONS

In keeping with long-standing patent law convention, the words “a” and“an” when used in the present specification in concert with the wordcomprising, including the claims, denote “one or more.” Some embodimentsof the invention may consist of or consist essentially of one or moreelements, method steps, and/or methods of the invention. It iscontemplated that any method or composition described herein can beimplemented with respect to any other method or composition describedherein.

As used herein, “therapeutically effective amount” refers to the amountof a compound that, when administered to a mammal for treating a state,disorder or condition, is sufficient to effect such treatment. The“therapeutically effective amount” will vary depending on the compound,the disease and its severity, and the age, weight, physical conditionand responsiveness of the mammal to be treated. As used herein the term“therapeutically effective amount” refers to an amount of a compoundsufficient to prevent, inhibit, reduce, or eliminate one or more causes,symptoms, or complications of elevated plasma levels of branched chainamino acids and/or branched chain alpha-ketoacid in an individual. Incertain embodiments, a desired therapeutic effect is the attainment oftarget plasma levels of branched chain amino acid and/or branched chainalpha-ketoacid for the individual. In specific embodiments, thetreatment is considered therapeutically effective when there is aparticular extent of reduction in the plasma level of one or morebranched chain amino acids and/or branched chain alpha-ketoacids. Incertain cases, the treatment is considered therapeutically effectivewhen there is a reduction of at least 5%, 7.5%, 10%, 12.5%, 15%, 17.5%,20%, 22.5%, 25%, 27.5%, 30%, 32.5%, 35%, 37.5%, 40%, 42.5%, 45%, 47.5%,or 50% of the plasma level of one or more branched chain amino acidsand/or branched chain alpha-ketoacids or when there is a reduction of atleast about 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%,30%, 32.5%, 35%, 37.5%, 40%, 42.5%, 45%, 47.5%, or 50% of the plasmalevel of one or more branched chain amino acids and/or branched chainalpha-ketoacids. The skilled artisan recognizes that plasma levels maybe measured by standard methods in the art, for example using a plasmaamino acid test or urine amino acid test by chromatography and/or massspectrometry.

As used herein, “treating” or “treatment” of a state, disorder orcondition includes: (1) preventing or delaying the appearance ofclinical symptoms of the state, disorder or condition developing in amammal that may be afflicted with or predisposed to the state, disorderor condition, but does not yet experience or display clinical orsubclinical symptoms of the state, disorder or condition; (2) inhibitingthe state, disorder or condition, i.e., arresting or reducing thedevelopment of the disease or at least one clinical or subclinicalsymptom thereof; or (3) relieving the disease, i.e., causing regressionof the state, disorder or condition or at least one of its clinical orsubclinical symptoms.

As used herein “ammonia scavenger drug” refers to drugs in the classwith phenylacetate (PAA) that function to bind glutamine or other aminoacids and result in the excretion of nitrogen. Thus, the term includesat least phenylbutyrate, phenylacetate, BUPHENYL®, AMMONUL®, AMMONAPS®,acetate, butyrate, butyroyloxymethyl-4-phenylbutyrate, glyceryltri-(4-phenylbutyrate), esters, ethers, and acceptable salts, acids, andderivatives thereof. Salts of 4-phenylbutyric acid may be of thealkaline or earth alkaline type, eg. lithium, sodium, potassium,magnesium or calcium, for example. In specific embodiments, the ammoniascavenger drug comprises histone deacetylase inhibitor activity.

II. MAPLE SYRUP URINE DISEASE

Treatment options for MSUD are extremely limited and generallyconstitute of dietary restriction of BCAA intake designed to maintainplasma levels within a range that is accepted to be nontoxic andsupports optimal growth and development. The recommended target dietarylevels of BCAA and thus the resulting plasma levels for diseasemanagement vary by age (Table 1). When prescribing diets based on “perkg body weight,” ideal weight (50^(th) percentile) for age is used tooptimize intake. This is most important when calculating diets forpatients who are failure-to-thrive. Strict dietary compliance isnecessary for effective management of the disease and to preventneurological damage.

TABLE 1 Maple Syrup Urine Disease 0 < 0.5 yrs 0.5 < 1 yrs 1 < 4 yrs 4 <7 yrs 7 < 11 yrs 11 < 19 yrs >19 yrs ILE, mg 75-35/kg 65-30/kg165-325/day 225-420/day 250-470/day 330-570/day 300-700/day * LEU, mg100-50/kg  75-35/kg 275-535/day 360-695/day 410-785/day 540-945/day400-1100/day   VAL, mg 80-40/kg 70-30/kg 200-375/day 250-500/day285-550/day 375-675/day 420-800/day * Protein, g 3.5-3.0/kg  3.0-2.5/kg   ≥30/day   ≥35/day   ≥40/day  50-65/day  50-65/day Energy, kcal100%-125% OF NAS/FNB RDA for age

The human BCKDC that is defective in MSUD is encoded by six genetic loci(E1α, E1β, E2, E3, BCKD kinase and BCKD phosphatase). Based on theaffected subunit of the human BCKDC, MSUD is classified into fourmolecular subgroups. These include types 1A, 1B, II, and III, referringto deficiencies in the E1α, E1β, E2, and the E3 subunits, respectively.A total of 166 MSUD mutations have been identified to date, with 50 intype 1A, 53 in type 1B, 49 in type 11, and 14 in type 111. BCKDCcatalyzes the rate-limiting oxidative decarboxylation of the branchedchain keto acids (BCKA) derived from the branched chain amino acids(BCAA), leucine, isoleucine, and valine. Mutations or deficiency in theenzyme activity results in accumulation of branched-chain 1-amino acids(BCAA) and branched chain keto acids (BCKA) that can exert neurotoxiceffects. MSUD presents as a heterogeneous clinical and molecularphenotype. Severity of the disease, ranging from classical to mildvariant types, is commonly classified on the basis of indirectparameters, e.g. onset, leucine tolerance and/or residual enzymeactivity in cells. Long-term therapy is based on dietary restriction ofBCAA intake designed to maintain plasma levels within range that isaccepted to be non-toxic and supports optimal growth and development(Table 2). The daily requirement of BCAA, in face of marked deficiencyof oxidative decarboxylation, is generally determined indirectly bymonitoring the effect of dietary treatment on growth.

TABLE 2 Branched Chain Target Levels Normal Reference Amino Acid(μmol/L) Range(μmol/L) Leucine 200-500 65-220 Isoleucine 100-200 26-100Valine 100-300 90-300

Since there is only one dehydrogenase enzyme for all three BCAAs, allthree α-keto acids accumulate and are excreted in the urine, giving itthe characteristic sweet smelling odor in afflicted individuals. Suchaccumulation can cause a variety of symptoms including lethargy,irritability, poor feeding, abnormal movements and a characteristic odorof maple syrup in the earwax (cerumen), sweat and urine of affectedindividuals.

MSUD is further confirmed by detection of elevated BCKAs, by gaschromatography-mass spectroscopic, analysis of urine, and elevated BCAAsin blood by amino acid analysis. Definitive diagnosis is established bylow measured activity of BCKDC in cultured lymphocytes or fibroblasts.

Severity of the disease, ranging from classical to mild variant types,is commonly classified on the basis of indirect parameters, e.g. onsetof symptoms, and dietary leucine tolerance and/or residual enzymeactivity in cells. There are five clinical subtypes of MSUD: the classicneonatal severe form, an intermediate form, an intermittent form, athiamine-responsive form, and an E3-deficient form with lactic acidosis.Traditionally, the metabolic phenotype is classified as classic orintermediate on the basis of residual BCKDC activity. Rarely, affectedindividuals have partial BCKDC deficiency that only manifestsintermittently or responds to dietary thiamine therapy. However,phenotypic distinctions are not absolute; individuals with intermediateor intermittent forms can experience severe metabolic intoxication andencephalopathy under sufficient catabolic stress. Moreover, in vitroassays of enzyme activity do not reliably predict clinical severity, ageof onset, or response to potential therapies.

In classic MSUD, which is the most common form of the disorder, 50% ormore of the ketoacids are derived from leucine, and the activity ofBCKDC is less than 2% of normal. Affected newborns appear normal atbirth, with symptoms developing between 4 to 7 days of birth. ClassicMSUD phenotype include maple syrup odor evident in creumen soon afterbirth and in urine five to seven days of age. In untreated neonates,ketonuria, irritability, and poor feeding is observed within 48 hours ofdelivery. Other classic symptoms like lethargy, intermittent apnea,opisthotonus, and stereotyped movements such as “fencing” and“bicycling” are evident by four to five days of birth. If left untreatedcoma and central respiratory failure follow. Additionally, following theneonatal period, acute leucine intoxication (leucinosis) andneurological deterioration can develop rapidly at an age as a result ofnet protein degradation precipitated by infection, surgery orpsychological stress. Each episode of leucinosis is associated with ahigh risk for cerebral edema. In older individuals, neurologicalsymptoms vary and may include cognitive impairment, hyperactivity,anorexia, sleep disturbances, hallucinations, mood swings, focaldystonia, choreoathetosis, and ataxia. In individuals of all agesdiagnosed with MSUD, nausea and vomiting are common during crisis andoften necessitate hospitalization. Increased plasma concentrations ofleucine and alpha-ketoisocaproic acid also lead to coma in variousindividuals.

Individuals with residual BCKDC activity, between 3-30% ex vivo, mayappear normal during the neonatal period but nevertheless have maplesyrup odor in cerumen and a consistently abnormal plasma amino acidprofile. These individuals may present with feeding problems, poorgrowth and developmental delays as infants, or may present much later inlife with nonsyndromic mental retardation. The majority of persons withintermediate MSUD are diagnosed between five months and seven years ofage. Severe leucinosis, brain swelling and death can occur ifindividuals with intermediate MSUD are subjected to severe catabolicstress. Basic management principles for these individuals are the sameas for those with classic MSUD, and the distinction between classic andintermediate type of MSUD is not absolute.

Intermittent MSUD is characterized by a normal growth and intellectualdevelopment throughout infancy and early childhood. Individuals normallycan tolerate normal leucine intake. Plasma amino acid and urine organicacid profiles for these individuals are normal or show a mild elevationof BCAAs. During infection or physiological stress, these individualsdevelop the clinical and biochemical features of classic MSUD, and inrare cases may progress to coma leading to death. BCKDC activity isroughly 5-20% of normal.

Thiamine-responsive MSUD was described as a variant of MSUD, in whichhyperaminoacidemia was completely corrected by thiamine hydrochloridewith dietary restriction. The existence of individuals presenting withtrue thiamine-responsive MSUD is not certain. In general, such putativeindividuals have a residual ex vivo BCKDC activity of up to 40% normaland are not ill in the neonatal period, but present later in life with aclinical manifestation similar to intermediate MSUD. Treatment involvesa combination of thiamine and dietary BCAA restriction. Hence, the invivo contribution of thiamine is not discernable.

E3-deficient MSUD with lactic acidosis or MSUD type III, presents acombined deficiency of the branched-chain alpha-keto acid dehydrogenase,pyruvate dehydrogenase, and alpha-keto glutarate dehydrogenasecomplexes.

Management of MSUD includes dietary leucine restriction, high-calorieBCAA-free formulas, and frequent monitoring (Tables 1 and 2). Metabolicdecompensation is corrected by treating the precipitating stress whiledelivering sufficient calories, insulin, free amino acids, isoleucineand valine, and in some cases hemodialysis/hemofilteration, to establishnet positive protein accretion. Brain edema is a common potentialcomplication of metabolic decompensation and requires immediate therapyin an intensive care setting. Adolescents and adults with MSUD and ADHD,depression, or anxiety respond to psychostimulants and anti-depressantmedications. Orthotopic liver transplant can be an effective albeit notoptimal therapy for classic MSUD. Frequent monitoring of plasma aminoacid concentrations and fetal growth maybe necessary to avoid essentialamino acid deficiencies during pregnancy.

III. GENERAL EMBODIMENTS OF THE INVENTION

Disorders resulting from inborn errors of metabolism (IEM) affect verysmall numbers of individuals, although the entire population of patientssuffering the results of inherited metabolic disorders is large. Singlegene defects result in abnormalities in the synthesis or catabolism ofproteins, carbohydrates, or fats. Most are due to a defect in an enzymeor transport protein, which results in a block in a metabolic pathway.Effects are due to toxic accumulations of substrates before the block,intermediates from alternative metabolic pathways, defects in energyproduction and use caused by a deficiency of products beyond the block,or a combination of these metabolic deviations. Nearly every metabolicdisease has several forms that vary in age of onset, clinical severity,and, often, mode of inheritance.

In certain embodiments of the invention, MSUD, is the result of aninborn error in metabolism that results in the accumulation of branchedchain amino acids and/or branched chain alpha-ketoacids. The inventionencompasses treatment of MSUD with an ammonia scavenger. In particularcases, a specific treatment regimen is employed. For example, particulardosages, formulations, schedule of administration, and so forth areutilized in the therapy of the individual.

Hence, disclosed herein is a method of decreasing plasma levels of atleast one of a branched chain amino acid and/or branched chainalpha-ketoacid comprising administering to an individual, in needthereof, a therapeutically effective amount of an ammonia scavengercompound or a pharmaceutically acceptable salt thereof, where the amountof the compound administered stimulates the baseline enzymatic activityof the branched chain dehydrogenase enzyme complex protein, to levelseffective in achieving target plasma levels of the branched chain aminoacid and/or branched chain alpha-ketoacid, for the individual. In someembodiments, “target plasma levels” of BCAA or BCKAs are levels thatachieve a therapeutic effect and are for example as defined in, but notlimited to, Table 2. A therapeutic effect refers to preventing,inhibiting, reducing, or eliminating one or more causes, symptoms, orcomplications of elevated plasma levels of branched chain amino acidsand/or branched chain alpha-ketoacid in an individual. The target plasmalevels of branched chain amino acid and/or branched chain alpha-ketoacidmay be varied depending on the age of the individual and the severity ofthe disease.

In general, the baseline enzymatic activity in MSUD individuals is about3% to about 30% of the normal activity of the branched chaindehydrogenase enzyme protein complex. Specifically, the baselineenzymatic activity is about 5% to about 20% of the normal activity ofthe branched chain dehydrogenase enzyme protein complex. In anembodiment, the baseline activity is about 0% to about 40%, about 2% toabout 40%, about 3% to about 30%, about 5% to about 20%, or 0% to about2% of the normal activity of the branched chain dehydrogenase enzymeprotein complex.

In specific embodiments of the invention, following administration ofthe therapeutic compositions of the invention, there is an increase ofbaseline enzymatic activity in MSUD individuals of at least 1% 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% over the levelof activity prior to the treatment.

In embodiments of the individual, the individual in need thereof is anindividual with an inborn error of metabolism. Specifically, the inbornerror of metabolism is the MSUD. Further, the MSUD is selected from agroup consisting of the intermediate form, the intermittent form or thethiamine-responsive form of the disease. In some cases, the branchedchain amino acid is at least one of leucine, isoleucine and valine.Moreover, in specific embodiments the branched chain alpha-ketoacid isat least one of keto-isocaproic acid, keto-methylvaleric acid, andketoisovaleric acid. In an embodiment, the ammonia scavenger compound isphenylbutyrate or a salt thereof. The salt is the sodium salt, calciumsalt, lithium salt or a potassium salt, in specific embodiments.Specifically, the phenylbutyrate or salt thereof causes decreasedphosphorylation of the S293 and S303 residues of the E1α subunit of thebranched chain dehydrogenase enzyme complex protein.

Additionally, disclosed herein in some embodiments is a method ofdecreasing plasma levels of at least one of a branched chain amino acidor branched chain alpha-ketoacid comprising administering to anindividual in need thereof a therapeutically effective amount of atleast one compound of the formula:

wherein n is 0, 2, 4, 6 or 8, or

a pharmaceutically acceptable salt or ester or prodrug thereof, wherethe amount of the compound administered stimulates the baselineenzymatic activity of the branched chain dehydrogenase enzyme complexprotein to levels effective in achieving target plasma levels ofbranched chain amino acid and/or branched chain alpha-ketoacid for theindividual.

In an embodiment, the baseline activity is about 0% to about 40%, about2% to about 40%, about 3% to about 30%, about 5% to about 20%, or 0% toabout 2% of the normal activity of the branched chain dehydrogenaseenzyme protein complex. In an embodiment, the compound is phenylbutyrateor a salt thereof. In a specific embodiment, the salt is sodium salt orthe calcium salt. Specifically, the phenylbutyrate or salt thereofcauses decreased phosphorylation of the S293 and S303 residues of the E1subunit of the branched chain dehydrogenase enzyme complex protein.Moreover, the branched chain amino acids are at least one of leucine,isoleucine and valine and the branched chain are at least one ofketo-isocaproic acid, keto-methylvaleric acid, and ketoisovaleric acid.In some embodiments, the compound may be administered orally,intra-peritoneally or intravenously. Further, the affected individual isan individual with high plasma levels of branched chain amino acidsand/or branched chain alpha-ketoacids as compared to those in a healthyindividual. Specifically, the high plasma levels of branched chain aminoacids and/or branched chain alpha-ketoacids are due to an inborn errorof metabolism resulting in MSUD.

Further, disclosed herein is a method for screening for a compounduseful for decreasing blood plasma levels of branched chain amino acidsand/or branched chain alpha-ketoacid by selecting or identifying acompound capable of decreasing the phosphorylation of the E1α subunit ofbranched chain dehydrogenase enzyme protein complex at position Ser293and Ser303, where the decrease in phosphorylation of the subunits at thepositions is effective in increasing the enzymatic activity of theenzyme from its baseline activity. The increase in the enzymaticactivity is defined as activity levels of the enzyme that are sufficientto catalyze the oxidative decarboxylation of branched chain amino acidor corresponding branched chain ketoacids, resulting in target plasmalevels of the same in an individual. In certain cases, the methodcomprises providing the E1α subunit of branched chain dehydrogenaseenzyme complex protein or a fragment thereof comprising at least 50consecutive amino acids that include phosphorylated Ser293 and Ser303residues or fragment thereof; contacting a candidate compound with theprotein or fragment thereof; and selecting the candidate compound thatcauses dephosphorylation of Ser293 and Ser303 of the E1α subunit ofbranched chain dehydrogenase enzyme complex. Furthermore, in someaspects the method comprises contacting a candidate compound with a cellexpressing the E1α subunit of branched chain dehydrogenase enzymecomplex protein and a branched chain amino acid dehydrogenase kinase;assessing the amount of E1α subunit phosphorylated at position S293 andS303; and selecting the candidate compound that decreases thephosphorylation of E1α subunit of branched chain dehydrogenase enzymecomplex in comparison with a control cell that has not been contactedwith the candidate compound.

Additionally disclosed herein in particular cases is a method oftreatment for MSUD comprising administering to an individual in needthereof a therapeutically effective amount of a compound or apharmaceutically acceptable salt thereof, where the amount of thecompound administered is effective in dephosphorylation of the S293 andS303 residues of the E1α subunit of branched chain dehydrogenase enzymecomplex protein thereby increasing the enzymatic activity of the enzymefrom baseline activity in the individual. Further, various methodscomprise restricting the dietary branched chain amino acid intake in theindividual. In general, the dephosphorylation is effective in increasingthe activity of the branched chain dehydrogenase enzyme complex protein.Specifically, the compound may be phenylbutyrate or a salt thereof. Thesalt is the sodium salt, calcium salt, lithium salt or a potassium salt,in certain aspects. The compound is administered orally,intra-peritoneally or intravenously. In general, the individual in needthereof is an individual with high plasma levels of the branched chainamino acids and/or branched chain alpha-ketoacids as compared to levelsof the respective branched chain amino acids and/or branched chainalpha-ketoacids in a healthy individual. Specifically, the individual isan individual having residual in vivo activity of the branched chaindehydrogenase enzyme complex protein. Further, the individual in need isan individual suffering from the classic form of MSUD. Alternatively,the individual is an individual with the intermediate form of MSUD. Incertain cases, the individual is an individual with the intermittentform of MSUD.

IV. PHARMACEUTICAL PREPARATIONS

Pharmaceutical preparations of the present invention are provided totreat individuals suffering from a medical condition resulting in aninborn error or metabolism. Particular pharmaceutical compositions ofthe present invention comprise an effective amount of one or moreammonia scavengers; in certain cases they are dissolved or dispersed ina pharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of a pharmaceutical composition thatcontains at least one ammonia scavenger will be known to those of skillin the art in light of the present disclosure, as exemplified byRemington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference. Moreover, for animal (e.g.,human) administration, it will be understood that preparations shouldmeet sterility, pyrogenicity, general safety and purity standards asrequired by FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the pharmaceuticalcompositions is contemplated.

The ammonia scavenger may comprise different types of carriers dependingon whether it is to be administered in solid, liquid or aerosol form,and whether it needs to be sterile for such routes of administration asinjection, for example. The present invention can be administeredintravenously, intradermally, transdermally, intrathecally,intraarterially, intraperitoneally, intranasally, intravaginally,intrarectally, topically, intramuscularly, subcutaneously, mucosally,nasally, intranasally, orally, topically, locally, inhalation (e.g.,aerosol inhalation), injection, infusion, continuous infusion, localizedperfusion bathing target cells directly, via a catheter, via a lavage,in cremes, in lipid compositions (e.g., liposomes), or by other methodor any combination of the forgoing as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

The ammonia scavenger may be formulated into a composition in a freebase, neutral or salt form. Pharmaceutically acceptable salts includethe acid addition salts, e.g., those formed with the free amino groupsof a proteinaceous composition, or which are formed with inorganic acidssuch as, for example, hydrochloric or phosphoric acids, or such organicacids as acetic, oxalic, tartaric or mandelic acid. Salts formed withthe free carboxyl groups can also be derived from inorganic bases suchas for example, sodium, potassium, ammonium, calcium or ferrichydroxides; or such organic bases as isopropylamine, trimethylamine,histidine or procaine. Upon formulation, solutions will be administeredin a manner compatible with the dosage formulation and in such amount asis therapeutically effective. The formulations are easily administeredin a variety of dosage forms such as formulated for parenteraladministrations such as injectable solutions, or aerosols for deliveryto the lungs, or formulated for alimentary administrations such as drugrelease capsules and the like.

Further in accordance with the present invention, the composition of thepresent invention suitable for administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, i.e.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent or carrier is detrimental to the recipient or to thetherapeutic effectiveness of the composition contained therein, its usein administrable composition for use in practicing the methods of thepresent invention is appropriate. Examples of carriers or diluentsinclude fats, oils, water, saline solutions, lipids, liposomes, resins,binders, fillers and the like, or combinations thereof. The compositionmay also comprise various antioxidants to retard oxidation of one ormore component. Additionally, the prevention of the action ofmicroorganisms can be brought about by preservatives such as variousantibacterial and antifungal agents, including but not limited toparabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol,sorbic acid, thimerosal or combinations thereof.

In accordance with the present invention, the composition is combinedwith the carrier in any convenient and practical manner, i.e., bysolution, suspension, emulsification, admixture, encapsulation,absorption and the like. Such procedures are routine for those skilledin the art.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach. Examples of stabilizers for use in an thecomposition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle compositions that include an ammoniascavenger, one or more lipids, and an aqueous solvent. As used herein,the term “lipid” will be defined to include any of a broad range ofsubstances that is characteristically insoluble in water and extractablewith an organic solvent. This broad class of compounds is well known tothose of skill in the art, and as the term “lipid” is used herein, it isnot limited to any particular structure. Examples include compounds thatcontain long-chain aliphatic hydrocarbons and their derivatives. A lipidmay be naturally occurring or synthetic (i.e., designed or produced byman). However, a lipid is usually a biological substance. Biologicallipids are well known in the art, and include for example, neutral fats,phospholipids, phosphoglycerides, steroids, terpenes, lysolipids,glycosphingolipids, glycolipids, sulphatides, lipids with ether andester-linked fatty acids and polymerizable lipids, and combinationsthereof. Of course, compounds other than those specifically describedherein that are understood by one of skill in the art as lipids are alsoencompassed by the compositions and methods of the present invention.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the ammonia scavenger may be dispersed in asolution containing a lipid, dissolved with a lipid, emulsified with alipid, mixed with a lipid, combined with a lipid, covalently bonded to alipid, contained as a suspension in a lipid, contained or complexed witha micelle or liposome, or otherwise associated with a lipid or lipidstructure by any means known to those of ordinary skill in the art. Thedispersion may or may not result in the formation of liposomes.

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount may vary according to the response of thesubject. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%,37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% of an active compound. In otherembodiments, the an active compound may comprise between about 1% toabout 90%, about 5% to about 90%, about 10% to about 80%, about 20% toabout 75%, about 25% to about 70%, about 30% to about 65%, about 35% toabout 60%, about 2% to about 75%, about 10% to about 50%, about 20% toabout 40%, about 25% to about 50%, about 5% to about 20%, about 50% toabout 75%, about 60% to about 80% or more of the weight of the unit, orbetween about 25% to about 60%, for example, and any range derivabletherein. Naturally, the amount of active compound(s) in eachtherapeutically useful composition may be prepared in such a way that asuitable dosage will be obtained in any given unit dose of the compound.Factors such as solubility, bioavailability, biological half-life, routeof administration, product shelf life, as well as other pharmacologicalconsiderations will be contemplated by one skilled in the art ofpreparing such pharmaceutical formulations, and as such, a variety ofdosages and treatment regimens may be desirable.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above. In certain cases, the amount that is used is atleast 250 mg/kg/day, at least 275 mg/kg/day, at least 300 mg/kg/day, atleast 325 mg/kg/day, at least 350 mg/kg/day, at least 375 mg/kg/day, atleast 400 mg/kg/day, at least 425 mg/kg/day, at least 450 mg/kg/day, atleast 475 mg/kg/day, at least 500 mg/kg/day, at least 525 mg/kg/day, atleast 550, at least 575 mg/kg/day, at least 600 mg/kg/day, at least 625mg/kg/day, at least 650 mg/kg/day, at least 675 mg/kg/day, at least 700mg/kg/day, at least 725 mg/kg/day, at least 750 mg/kg/day, at least 775mg/kg/day, at least 800 mg/kg/day, at least 825 mg/kg/day, at least 850mg/kg/day, at least 875 mg/kg/day, at least 900 mg/kg/day, at least 925mg/kg/day, at least 950 mg/kg/day, at least 975 mg/kg/day, at least 1000mg/kg/day, at least 1.25 g/kg/day, at least 1.5 g/kg/day, at least 1.75g/kg/day, at least 2 g/kg/day, at least 2.5 g/kg/day, at least 3g/kg/day, at least 3.5 g/kg/day, at least 4 g/kg/day, at least 4.5g/kg/day, at least 5 g/kg/day, at least 5.5 g/kg/day, at least 6g/kg/day, at least 6.5 g/kg/day, at least 7 g/kg/day, at least 7.5g/kg/day, or at least 10 g/kg/day. In specific embodiments, a dosage of450-600 mg/kg/day is administered.

A. Alimentary Compositions and Formulations

In particular embodiments of the present invention, the ammoniascavenger is formulated to be administered via an alimentary route.Alimentary routes include all possible routes of administration in whichthe composition is in direct contact with the alimentary tract.Specifically, the pharmaceutical compositions disclosed herein may beadministered orally, buccally, rectally, or sublingually, for example.As such, these compositions may be formulated with an inert diluent orwith an assimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

In certain embodiments, the active compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tables,troches, capsules, elixirs, suspensions, syrups, wafers, and the like(Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515;5,580,579 and 5,792,451, each specifically incorporated herein byreference in its entirety). The tablets, troches, pills, capsules andthe like may also contain the following: a binder, such as, for example,gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; anexcipient, such as, for example, dicalcium phosphate, mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate or combinations thereof; a disintegrating agent, such as, forexample, corn starch, potato starch, alginic acid or combinationsthereof; a lubricant, such as, for example, magnesium stearate; asweetening agent, such as, for example, sucrose, lactose, saccharin orcombinations thereof; a flavoring agent, such as, for examplepeppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.When the dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar, or both. When the dosage form is a capsule, it maycontain, in addition to materials of the above type, carriers such as aliquid carrier. Gelatin capsules, tablets, or pills may be entericallycoated. Enteric coatings prevent denaturation of the composition in thestomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No.5,629,001. Upon reaching the small intestines, the basic pH thereindissolves the coating and permits the composition to be released andabsorbed by specialized cells, e.g., epithelial enterocytes and Peyer'spatch M cells. A syrup of elixir may contain the active compound sucroseas a sweetening agent methyl and propylparabens as preservatives, a dyeand flavoring, such as cherry or orange flavor. Of course, any materialused in preparing any dosage unit form should be pharmaceutically pureand substantially non-toxic in the amounts employed. In addition, theactive compounds may be incorporated into sustained-release preparationand formulations.

For oral administration, the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation, for example. For example, a mouthwashmay be prepared incorporating the active ingredient in the requiredamount in an appropriate solvent, such as a sodium borate solution(Dobell's Solution). Alternatively, the active ingredient may beincorporated into an oral solution such as one containing sodium borate,glycerin and potassium bicarbonate, or dispersed in a dentifrice, oradded in a therapeutically-effective amount to a composition that mayinclude water, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively, the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

Additional formulations that are suitable for other modes of alimentaryadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum. After insertion, suppositories soften, melt or dissolvein the cavity fluids. In general, for suppositories, traditionalcarriers may include, for example, polyalkylene glycols, triglyceridesor combinations thereof. In certain embodiments, suppositories may beformed from mixtures containing, for example, the active ingredient inthe range of about 0.5% to about 10%, or about 1% to about 2%, forexample.

B. Parenteral Compositions and Formulations

In further embodiments, an ammonia scavenger may be administered via aparenteral route. As used herein, the term “parenteral” includes routesthat bypass the alimentary tract. Specifically, the pharmaceuticalcompositions disclosed herein may be administered for example but notlimited to intravenously, intradermally, intramuscularly,intraarterially, intrathecally, subcutaneous, or intraperitoneally (seeU.S. Pat. Nos. 6,613,308; 5,466,468; 5,543,158; 5,641,515; and5,399,363, for example (each specifically incorporated herein byreference in its entirety).

Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofor in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy injectability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (i.e., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in isotonic NaCl solution andeither added hypodermoclysis fluid or injected at the proposed site ofinfusion, (see for example, “Remington's Pharmaceutical Sciences” 15thEdition, pages 1035-1038 and 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. A powdered composition is combined with a liquidcarrier such as, e.g., water or a saline solution, with or without astabilizing agent.

C. Miscellaneous Pharmaceutical Compositions and Formulations

In other preferred embodiments of the invention, the active compoundammonia scavenger may be formulated for administration via variousmiscellaneous routes, for example, topical (i.e., transdermal)administration, mucosal administration (intranasal, vaginal, etc.)and/or inhalation.

Pharmaceutical compositions for topical administration may include theactive compound formulated for a medicated application such as anointment, paste, cream or powder. Ointments include all oleaginous,adsorption, emulsion and water-solubly based compositions for topicalapplication, while creams and lotions are those compositions thatinclude an emulsion base only. Topically administered medications maycontain a penetration enhancer to facilitate adsorption of the activeingredients through the skin. Suitable penetration enhancers includeglycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones andluarocapram. Possible bases for compositions for topical applicationinclude polyethylene glycol, lanolin, cold cream and petrolatum as wellas any other suitable absorption, emulsion or water-soluble ointmentbase. Topical preparations may also include emulsifiers, gelling agents,and antimicrobial preservatives as necessary to preserve the activeingredient and provide for a homogenous mixture. Transdermaladministration of the present invention may also comprise the use of a“patch”. For example, the patch may supply one or more active substancesat a predetermined rate and in a continuous manner over a fixed periodof time.

In certain embodiments, the pharmaceutical compositions may be deliveredby eye drops, intranasal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering compositions directly to thelungs via nasal aerosol sprays has been described e.g., in U.S. Pat.Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein byreference in its entirety). Likewise, the delivery of drugs usingintranasal microparticle resins (Takenaga et al., 1998) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,specifically incorporated herein by reference in its entirety) are alsowell-known in the pharmaceutical arts. Likewise, transmucosal drugdelivery in the form of a polytetrafluoroetheylene support matrix isdescribed in U.S. Pat. No. 5,780,045 (specifically incorporated hereinby reference in its entirety).

The term aerosol refers to a colloidal system of finely divided solid ofliquid particles dispersed in a liquefied or pressurized gas propellant.The typical aerosol of the present invention for inhalation will consistof a suspension of active ingredients in liquid propellant or a mixtureof liquid propellant and a suitable solvent. Suitable propellantsinclude hydrocarbons and hydrocarbon ethers. Suitable containers willvary according to the pressure requirements of the propellant.Administration of the aerosol will vary according to subject's age,weight and the severity and response of the symptoms.

The compounds of the present invention can be used separately or in theform of mixtures, including mixtures of acids and/or salts. Thecompounds of the present invention may be in the forms normallyemployed, such as tablets, capsules, powders, syrups, solutions,suspensions and the like, and may contain flavorants, sweeteners, etc.,in suitable solid or liquid carriers or diluents, or in suitable sterilemedia to form injectable solutions or suspensions. The active compoundsof the invention will be present in such pharmaceutical compositions inthe amounts sufficient to provide the desired dosage in the range asdescribed above. Thus, for oral administration, the compounds can becombined with a suitable solid, liquid carrier or diluent to formcapsules, tablets, powders, syrups, solutions, suspensions and the like.The pharmaceutical compositions, may, if desired, contain additionalcomponents such as flavorants, sweeteners, excipients and the like. Forparenteral administration, the compounds can be combined with sterileaqueous or organic media to form injectable solutions or suspensions.For example, solutions in aqueous propylene glycol and the like can beused, as well as aqueous solutions of water-solublepharmaceutically-acceptable acid addition salts or salts with base ofthe compounds of the present invention can be used. The injectablesolutions prepared in this manner can then be administeredintravenously, intraperitoneally, subcutaneously, or intramuscularly,with intramuscular administration being preferred in humans. In someembodiments, the formulation is supersaturated, include taste masking,and/or encompass liquid concentrated formulations, for example.

The compounds in accordance with the present invention may be formulatedinto various unit dosage forms such as tablets, soft and hard capsules,solutions and the like, by addition of pharmaceutically acceptablecarriers, for example diluents such as lactose, lubricants such asmagnesium stearate, binding agents such as polyvinylpyrrolidone anddisintegrating agents such as calcium carboxymethyl-cellulose. Prolongedrelease of the active compounds in accordance with the present inventionis also contemplated and is to be understood as being especially a rateof release of the active ingredient over a period of about 6 to 12 or upto 24 hours, for example.

Additionally, a dose of the pharmaceutical composition in accordancewith the present invention can be appropriately determined dependingupon various factors such as age and symptoms of patients, dosage formsand kinds of drugs. The size and frequency of the dosages given at anytime may be varied as desired provided the indicated total daily dose isnot significantly modified. Even though a unit dose of the compounds inaccordance with the present invention varies depending upon variousfactors such as severity of disease and age of individual, it may begenerally in the range of 8-13 grams/m²/day mg, and preferably 10grams/m²/day. As used herein, the term “unit dose” refers to a dailydose of the compounds of the present invention for an individual whichmay be administered singly or as a divided dose once or several times aday. The compounds in accordance with the present invention arepreferably given orally by administration of the unit dose as a singledose or divided dose once to four times a day. However, dosage amountsand frequency of administration may vary.

D. Specific Exemplary Formulations

In certain embodiments of the invention, there is a novel therapeuticuse for am ammonia scavenger as an effective modulator of plasma levelsof branched chain amino acids and/or branched chain alpha-ketoacids andtheir metabolites for the effective treatment of MSUD, and in some casesin combination with a restriction of dietary intake of branched chainamino acids and/or branched chain alpha-ketoacids, in specific cases.

In certain embodiments of the invention, there is a novel therapeuticuse for sodium phenylbutyrate and its esters or prodrugs or derivativesas an effective modulator of plasma levels of branched chain amino acidsand/or branched chain alpha-ketoacids and their metabolites for theeffective treatment of MSUD, and in some cases in combination with arestriction of dietary intake of branched chain amino acids and/orbranched chain alpha-ketoacids, in particular aspects.

In specific embodiments of the invention, the ammonia scavengercomprises phenylbutyrate (PBA) and in particular cases is used as atablet or a powder. PBA has multiple biological activities, includingits well known histone deacetylase (HDAC) inhibition activity, chemicalchaperoning upon endoplasmic reticulum (ER) stress, and ammoniascavenging in urea cycle dysfunction. Sodium phenylbutyrate is apro-drug and is rapidly metabolized to phenylacetate. Phenylacetate, ametabolically active compound, is first activated to its co-enzyme Aester, and then converted to phenylacetylCoA via beta-oxidationconjugates with glutamine via acetylation to form phenylacetylglutaminein the liver and kidneys. Phenylacetylglutamine is then excreted by thekidneys, providing an alternate vehicle for nitrogen excretion. On amolar basis, it is comparable to urea (each containing two moles ofnitrogen). Therefore, phenylacetylglutamine provides an alternatevehicle for waste nitrogen excretion by increasing glutamine clearance.

In certain embodiments, the present invention discloses a novel use ofthe well known chemical compound phenylbutyrate, as a therapeuticallyeffective modulator of plasma levels of branched chain amino acidsand/or branched chain alpha-ketoacids and their metabolites. Withoutwishing to be bound by any theory, it is believed that the mechanism ofBCAA depression by sodium phenylbutyrate may arise via two differentpossibilities. The first mechanism of PBA action may exploit its knownactivity as a Histone deacetylase (HDAC) inhibitor, whereby it affectsthe transcriptional levels of the target protein, the BCKDC. A secondnovel mechanism of action of PBA disclosed herein is its effects on thephosphorylation status of BCKDC, which is effective in increasing theenzymatic activity of the branched chain dehydrogenase enzyme complexprotein.

Certain ammonia scavengers may be employed in the invention, includingthose from U.S. Patent Application Publication US2010/0008859, which isincorporated by reference herein in its entirety. In particularembodiments of the invention, Buphenyl®/Ammonaps tablets are utilized.In other embodiments of the invention, Buphenyl®/Ammonaps powder areutilized. The skilled artisan recognizes that each tablet of Buphenyl®contains 500 mg of sodium phenylbutyrate and the inactive ingredientsmicrocrystalline cellulose NF, magnesium stearate NF, and colloidalsilicon dioxide NF, and also that each gram of Buphenyl® Powder contains0.94 grams of sodium phenylbutyrate and the inactive ingredients calciumstearate NF, and colloidal silicon dioxide NF; such a composition may beemployed in the invention.

In some cases, HPN-100 is administered to the individual. In certaincases, a formulation is prepared and/or administered as described in PCTPublication WO/2008/083226 and WO 2007005633, which are incorporatedherein by reference in their entirety.

In specific cases, there are preparations of high liquid dosage of anammonia scavenger (for example, sodium 4-phenylbutyrate) in aconcentrated aqueous composition, in some cases comprising at least oneof a preservative and a sweetening agent, and in specific cases both, inaddition to a flavoring agent. In certain embodiments, a fragrance canalso be added. The supersaturated composition can have a concentrationup to 500 mg/mL of sodium 4-phenylbutyrate or more, for example, such asthe concentration ranges from about 300 mg/mL to about 700 mg/mL. Apreservative such as sodium benzoate can be present, such as at about2.5 mg/mL, in specific aspects. In other embodiments, the dosage caninclude a sweetening and/or other flavoring agent, such as about 2 mg/mLof sodium saccharine or 0.01 mg/mL of sucralose. In some embodiments aflavoring agent such as about 2 mg/mL of flavoring. This highlyconcentrated liquid dosage is more concentrated and more palatable,leading to easier administration to young patients and facilitatingimproved compliance to the dosing regimen. This concentrated solution iseffective and very easy to administer to babies, because it requiresonly a few milliliters at any one dosing time and it is easy toadminister to children because each dosage is only a few milliliters ofsolution at any one time.

The skilled artisan recognizes that there is a process of preparing asupersaturated composition of sodium 4-phenylbutyrate, for example, inwater by adding sufficient water to a known quantity of sodium4-phenylbutyrate at an elevated temperature of about 30° to about 800°C. to produce a concentration of about 600 mg/mL, or by adding thecompound to a known quantity of water. The composition can be adjustedto a different pH, such as with an acid such as hydrochloric acid, forexample. In other embodiments, there is a process for making4-phenylbutyrate from 4-phenylbutyric acid by dissolving the same in anorganic medium, treating with an inorganic alkali, heating, adding asecond solvent to precipitate the product, and isolating/purifying theproduct.

Yet another object of this invention is to provide a process formanufacturing sodium 4-phenylbutyrate, for example, with impurities at alevel less than 0.05% (weight/weight basis). The general processprovided by this invention is to treat Ph-(CHi)₂-CH(COOEt)₂ (i.e.,diethyl 2-phenylethylmalonate) with acetic acid and aqueous hydrochloricacid to produce A-phenylbutyric (or 4-phenylbutanoic) acid. In anotherembodiment, conversion of 4-phenylbutyric acid to its sodium salt isaccomplished in an organic solvent medium with an inorganic base. Inspecific embodiments this invention provides a pharmaceutical liquidcomposition comprising a solution of sodium 4-phenylbutyrate in anaqueous medium at a concentration of at least about 300 mg/mL, includinggenerally at a concentration of about 300 mg/mL to about 700 mg/mL, andmore preferably at a concentration of about 400 mg/mL to about 600mg/mL, for example. As a dosage the composition preferably furthercomprises at least one or more of a flavoring agent, includingsweeteners, a preservative, and compatible mixtures thereof. Thecomposition may also include an inorganic base.

In certain embodiments, the composition administered to the patientcomprises a preservative, a flavoring agent, a fragrance, or a mixturethereof. The composition can also further comprise a preservative and aflavoring agent. The composition can also further comprise a fragranceand a sweetener as the flavoring agent.

In one embodiment, a pharmaceutical liquid composition is providedcomprising sodium 4-phenylbutyrate in an aqueous medium at aconcentration of at least about 300 mg/mL. In certain embodiments, thecomposition further comprising a preservative. The composition can alsofurther comprise a flavoring agent. In certain embodiments, thecomposition comprises both a preservative and a flavor. In someembodiments, the composition comprises at least two flavoring agents anda preservative. The composition can include sodium 4-phenylbutyrate at aconcentration range from about 300 mg/mL to about 700 mg/mL. Thecomposition can also contain sodium 4-phenylbutyrate in the range fromabout 400 mg/mL to about 600 mg/mL. The composition can also contain thecompound at a concentration of about 500 mg/mL. In certain embodiments,the weight fraction of water is less than the weight fraction of sodium4-phenylbutyrate.

The preservative can be sodium benzoate, in certain cases. In certainembodiments, the sweetening agent is sodium saccharine. In otherembodiments, the sweetening agent is sucralose. The composition cancomprise a mixture of sodium saccharine and sucralose.

The composition can also further comprise a base. In certainembodiments, the base is sodium carbonate. The base can also be sodiumhydroxide. The composition can further comprise 4-phenylbutyric acid.The composition can also further comprise sodium carbonate. In someembodiments, the aqueous composition does not freeze at 0° C.

Regarding sodium phenylbutyrate as an exemplary ammonia scavenger, it isknown that in some cases peak plasma levels of phenylbutyrate occurwithin 1 hour after a single dose of 5 grams of sodium phenylbutyratetablet with a C_(max) of 218 μg/mL under fasting conditions; peak plasmalevels of phenylbutyrate occur within 1 hour after a single dose of 5grams of sodium phenylbutyrate powder with a C_(max) of 195 μg/mL underfasting conditions. Buphenyl® is combined with dietary proteinrestriction and, in some cases, essential amino acid supplementation.Each Buphenyl® Tablet contains 62 mg of sodium (9.2% w/w) (correspondingto 124 mg of sodium per gram of sodium phenylbutyrate [12.4% w/w]) andBuphenyl® Powder contains 11.7 grams of sodium per 100 grams of powder,corresponding to 125 mg of sodium per gram of sodium phenylbutyrate(12.4% w/w).

In particular embodiments of the invention, 450-600 mg/kg/day of sodiumphenylbutyrate is given in neonates, infants and children weighing lessthan 20 kg, and 9.9-13.0 g/m²/day is given in children weighing morethan 20 kg, including adolescents and adults. The skilled artisanrecognizes that one mole of sodium phenylbutyrate is metabolized to onemole of phenylacetylglutamine, and from the estimated nitrogen to beexcreted on a restricted intake. Excretion of 0.09 g/kg/d ofphenylacetylglutamine nitrogen would require a dose of 0.6 g/kg/d ofsodium phenylbutyrate, in certain cases.

Based on pharmacokinetic data of phenylbutyrate in humans as well as invitro kinetic studies of phenylbutyrate on the BCKD complex, a specificdosing regimen is required to achieve plasma levels sufficient foraltering the phosphorylation of E1α of the BCKD complex. Based on invitro cell, recombinant protein, and human PK analysis, at least 1 mMC_(max) in plasma is required to achieve this effect. To achieve this, adosing regimen of sodium phenylbutyrate of 10 grams/m² surface area/daydivided either three times per day or more would be needed. This dosingshould be modified based on the PK properties of other formulations orderivatives of phenylbutyrate.

AMMONAPS may be administered to the individual, in some cases with aprotein-reduced diet. In certain cases, it is administered by mouth,through a gastrostomy, or through a nasogastric tube and, in some cases,with each meal or feeding. In certain cases, at least three hours shouldpass before a subsequent dose. The skilled artisan recognizes thatAMMONAPS comprises sodium phenylbutyrate and that in 1 g there are 940mg of sodium phenylbutyrate; other ingredients include calcium stearateand colloidal anhydrous silica. In order to dose accurately andespecially for smaller amounts required for infants, three measuringspoons are utilized for the granules, giving doses of 0.95 g, 2.9 g and8.6 g.

Pharmacokinetics after oral administration of phenylbutyrate have beenstudied in healthy volunteers (single dose of 2.5 g, n=2; single dose of5 g, n=21), in one patient with ornithine transcarbamylase deficiencyand in 8 patients with haemoglobinopathies. Phenylbutyrate is rapidlyabsorbed: measurable plasma levels of phenylbutyrate are detected 15 minafter oral administration. Peak concentrations of approximately 1 mmol/1are reached after 1 h. In one study, the elimination half-life wasestimated to be 0.8 h. Measurable plasma levels of phenylacetate (PA)and phenylacetateglutamine (PAG) are detected 30-60 min after oraldosing of phenylbutyrate (the mean peak concentration is 45.3 and 62.8μg/ml, respectively). The time to peak concentration increases with thedose of PB and is around 3.5 h for both metabolites after a dose of 5 gof phenylbutyrate. The elimination half-life was estimated to be 1.3 and2.4 hours, respectively for PA and PAG. Recovery of phenylbutyrate andPAG from serial collections of urine has been evaluated in some of thecited studies. It is demonstrated that in most subjects, the kidneyswithin 24h excrete approximately 80-100% of the drug as the conjugatedproduct, PAG.

V. NUTRITIONAL MANAGEMENT

In certain embodiments of the invention, the individual is subject todietary restriction in addition to utilization of the methods andcompositions of the invention. To promote growth and development ininfants and children, plasma levels of branched-chain amino acids and/orbranched chain alpha-ketoacids are carefully monitored; levels in adultsare also monitored. In individuals with MSUD, a diet with minimal levelsof the amino acids leucine, isoleucine, and valine must be maintained inorder to prevent neurological damage. When the condition is diagnosed,and during episodes, treatment may include eating a protein-free diet.Fluids, sugars, and possibly fats may be given through a vein (IV).Peritoneal dialysis or hemodialysis can be used to reduce the level ofcertain amino acids.

The health care provider will follow the levels of the branched chainamino acids and/or branched chain alpha-ketoacids closely and willadjust the diet based on the amino acid levels. Long term treatmentrequires a special diet, and the diet in infants may include a man-madeinfant formula with low levels of the amino acids leucine, isoleucine,and valine. Persons with this condition may remain on this dietpermanently.

In certain embodiments of the invention, the diet of the individual withMSUD includes a variety of general factors. Intake of the branched aminoacids, which are essential, must be carefully monitored. For example,the individual's tolerance of leucine must be calculated followingmeasurement of BCAA levels and re-measured at appropriate intervalsduring about the first 6 to 12 months of life. The individual may intakea protein substitute that provides BCAA-free amino acids, in certaincases. In particular aspects, the individual intakes a supplement thatprovides necessary vitamins, minerals, and trace elements. In somecases, isoleucine, leucine, and/or valine supplements, taken as needed.In some cases the patient's levels of isoleucine and valine fall belowdesirable levels, or are too low in reference to the leucine level.

The individual may consume an adequate intake of calories from one ormore of foods naturally low in or free from protein; speciallyformulated low-protein foods; and protein-free energy supplementscontaining glucose polymers and fats, in certain aspects of theinvention.

Infants diagnosed with MSUD may be administered a special MSUD formulasupplemented with controlled amounts of infant formula. Breastfeeding isbeneficial to some children with MSUD but does not remove the need forthe special formula. From childhood to the age of 10, the individualmust continue to take a protein substitute along with other foods thatare monitored to supply the correct amount of leucine. BCAA levelsshould be re-evaluated at least every 6-12 months. In individuals thatare over the age of about 8 years old, the protein substitute maycontain a certain amount of a protein equivalent (for example, 10 grams,12 grams, 15 grams, 17 grams, or 20 grams) and may be taken as alow-volume drink. The mixture may be supplied as a powder that containsthe daily requirements of amino acids, vitamins, minerals, and/or traceelements. As with children, adolescent and adult patients should havetheir leucine levels measured periodically.

At the recommended dose of sodium phenylbutyrate, for example, it issuggested that infants with neonatal-onset CPS and OTC deficienciesinitially receive a daily dietary protein intake limited toapproximately 1.6 g/kg/day for the first 4 months of life. If tolerated,the daily protein intake may be increased to 1.9 g/kg/day during thisperiod. Protein tolerance will decrease as the growth rate decreases,requiring a reduction in dietary nitrogen intake. From 4 months to 1year of age, it is recommended that the infant receive at least 1.4g/kg/day, but 1.7 g/kg/day is advisable. From 1 to 3 years of age, theprotein intake should not be less than 1.2 g/kg/day; 1.4 g/kg/day isadvisable during this period. If caloric supplementation is indicated, aprotein-free product is recommended. Caloric intake should be based uponthe “Recommended Dietary Allowances”, 10th ed., Food and NutritionBoard, National Research Council, National Academy of Sciences, 1989.

VI. KITS OF THE INVENTION

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, an ammonia scavenger may be comprised in a kit.The kits will thus comprise, in suitable container means, an ammoniascavenger and, optionally, a diet composition with restricted branchchain amino acids of the present invention. The components of the kitmay be packaged either in aqueous media or in lyophilized form. Thecontainer means of the kit will generally include at least one vial,test tube, flask, bottle, syringe or other container means, into which acomponent may be placed, and preferably, suitably aliquoted. Where thereare more than one component in the kit, the kit also will generallycontain a second, third or other additional container into which theadditional components may be separately placed. However, variouscombinations of components may be comprised in a vial. The kits of thepresent invention also will typically include a means for containing theammonia scavenger and any other reagent containers in close confinementfor commercial sale. Such containers may include injection or blowmolded plastic containers into which the desired vials are retained.

The ammonia scavenger may be formulated into a syringeable composition.In which case, the container means may itself be a syringe, pipette,and/or other such like apparatus, from which the formulation may beapplied to an infected area of the body, injected into an animal, and/oreven applied to and/or mixed with the other components of the kit.

However, the components of the kit may be provided as dried powder(s).When reagents and/or components are provided as a dry powder, the powdercan be reconstituted by the addition of a suitable solvent. It isenvisioned that the solvent may also be provided in another containermeans.

VII. EXAMPLES

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way,however, be construed as limiting the broad scope of the invention.

Example 1 Clinical Protocol

The clinical protocol was approved by the Human Subjects InstitutionalReview Board of the Baylor College of Medicine. The healthy controlsubjects and the MSUD patients were admitted into the Texas Children'sHospital General Clinical Research Center and were started on the studyprotocol after an informed consent was obtained. Each subject or aparent for those younger than 18 years gave written informed consent forparticipation in the study. Both the healthy controls (N=3) and the MSUDpatients (N=5) were admitted twice in the clinical research center for 3days each. For both admissions, the subjects received a constant proteinintake of 0.6 grams/kg/day as a combination of BCAA-free formula andwhole protein. On day three of admission, the patient had blood samplingat 0, 4, 6, and 8 hours during a period of frequent every two hour feedsin which ⅛th of the day's protein subscription was given. On the secondadmission, each subject was given sodium phenylbutyrate (Buphenyl) at adose of 10 grams/m²/day divided into four equal doses. Otherwise, bloodsampling was performed in the fed state on day 3 as in the baselineadmission. Plasma samples were analyzed for amino acids and theircorresponding BCKA: α-keto-β-methylvalerate (KMV), α-ketoisocaproate(KIC), and α-ketoisovalerate (KIV). The concentrations of the plasmaamino acids were measured with the amino acid analyzer method. PlasmaBCKA were derivatized with o-phenylenediamine and separation was made bygradient elution from a Spherisorb™ ODS2 column (250 mm×4.6 mm, 5 μm;Waters) according to protocols previously described.

PBA Treatment Causes a Decrease in the BCAA and the BCKA Levels

In distinguishing the mechanism of BCAA depression by sodiumphenylbutyrate, BCAA and BCKA levels were measured in control healthysubjects on a steady state protein intake, before and after sodiumphenylbutyrate administration. These control subjects formed a part of alarger study comparing the efficacy of sodium phenylbutyrate vs. sodiumbenzoate to decrease ureagenesis. BCAA (FIG. 1) and BCKA (FIG. 2) plasmalevels were found to be reduced in control subjects treated with sodiumphenylbutyrate (FIGS. 1 and 2).

Administration of PBA Correlates with Decreased Levels of BCAA and BCKAExpression in MSUD Patients

To determine whether the BCAA and BCKA depression by sodiumphenylbutyrate may be of therapeutic benefit in MSUD, plasma BCAA andBCKA levels were measured in MSUD subjects, on a steady state proteinintake, before and after sodium phenylbutyrate administration. BCAA(FIG. 3) levels were found to be reduced and BCKA (FIG. 4) levels werefound to be significantly decreased in three of five MSUD subjectstreated with sodium phenylbutyrate (FIGS. 3 and 4). In the three MSUDpatients, that demonstrated a decrease in plasma BCAA and BCKA levels,in response to sodium phenylbutyrate administration, leucine reductionranged from 24% to 34% of the baseline levels. There was no clearcorrelation between the levels of residual activity or the mutatedsubunit of the BCKD complex and the biochemical response tophenylbutyrate.

Example 2

Measurement of BCKDC Expression and Activity in Patients withIntermediate and Late Onset of MSUD

Five patients with intermediate and/or late onset forms of MSUD wererecruited for a trial with phenylbutyrate. Patients with theintermediate form have some degree of residual enzyme activity, and,therefore, were hypothesized to have higher likelihood to respond to thetreatment as compared to patients with the classic form who have noresidual activity. Diagnosis of the intermediate form was made based onclinical onset of clinical symptoms beyond the neonatal period. Thediagnosis of MSUD was confirmed biochemically based on the elevatedleucine and on the presence of alloisoleucine in plasma. Enzyme assayand DNA analysis for BCKDC activity and genotype, respectively, on thesesubjects were performed and summarized (Table 3). BCKDC enzyme activitywas measured on skin fibroblasts obtained from all five patients usingradioactive method previously described. In this method, culturedfibroblasts cells are incubated with α-1-¹⁴C-leucine for 4 hours in thepresence of 1 mM α-chloroisocaproate in the medium to stimulate theBCKDC activity. At the end of the incubation, the amount of ¹⁴CO2released from leucine decarboxylation is captured onto damped filterpaper. Decarboxylation activity of BCKDC is expressed as pmol of CO₂released/mg protein/hour and percentage of normal activity.

DNA samples from the five patients were analyzed for mutations in theBCKDHA, BCKDHB, and DBT genes by sequencing all the coding exons andtheir flanking intronic regions. When only one mutation was found bysequencing, the DNA samples were further analyzed by targeted array CGHto rule out microdeletions. Interestingly, the in vitro enzyme activitydid not correlate with the clinical presentation because three of thefive patients have very low activity (<5%) despite the late-onset of thedisease.

TABLE 3 BCKD activity* DNA analysis % of Affected Age Mean ± normalenzyme (years) Gender SD control subunit Allele 1 Allele 2 Patient 1(AG) 24 M   3 ± 2.81 0.96 Pending Pending Pending Patient 2 (JK) 17 M7.4 ± 6.7 0.9 E1α c.887_894del p.Y393N Patient 3 (CR) 5 F   2 ± 0.740.26 E1α p.V412M p.V412M Patient 4 (GP) 6 F 272 ± 31  36.1 E2 c.75_76delp.R301C Patient 5 (CG) 16 F 7 ± 1 1.62 E2 p.S366P Exon 11 del *pmol CO2released/mg protein/hour

Example 3 PBA Treatment Specifically Increased BCKDC Activity

Table 4 shows the effects of Phenylbutyrate on Control and MSUDfibroblast cells. To confirm that the effect of the phenylbutyrate wasspecific for BCKD activity, the enzyme activity was measured in controland MSUD patient fibroblasts before and after incubation withphenylbutyrate at the concentration of 2 mM from two independentpatients (one clinical responder and one clinical nonresponder) and inone control. The control fibroblast cell line showed a 1.7 fold increasein enzyme activity after incubation with phenylbutyrate. A similarincrease (1.7 fold) over baseline activity was also observed in one MSUDcell line (patient 5), consistent with the biochemical response for BCAAand α-ketoacids in that patient. However, fibroblasts from Patient 3 didnot show an increase of enzyme activity over baseline levels.

TABLE 4 Phenylbutyrate − + Normal control  100% 176.08% Patient 3(CR)-non 0.59%  0.52% responder clinically Patient 5 (CG)- 4.47%  7.62%responder clinically

Example 4 PBA Treatment Decreases the Phosphorylation of the E1-AlphaSubunit of the BCKDC

A post-translational mechanism of regulation of the activity of BCKDC bycovalent modification by phosphorylation. In order to investigate theeffect and mechanism of phenylbutyrate-mediated increased activity ofBCKDC, Buphenyl® or saline were given orally to C57B6 mice (N=5 mice pergroup) by gavage at the dose of 50 mg/kg/day divided into 3administrations for 3 consecutive days. After three days of treatmentthe animals were sacrificed to harvest the liver samples. Proteins wereextracted from mouse livers by homogenizing the tissue in a buffercontaining 5% SDS and 0.0625 M TrisHCl. Western blot analyses wereperformed using an anti-Phospho BCKDC antibody (gift from Dr. Lynch),the anti-BCKDC-E1 antibody (21), and the anti-BCKDC-E2 antibody (KamiyaBiomedical Company). The Western blot analysis on the liver extractshowed that the phenylbutyrate (PB A) treatment resulted in asignificant reduction of the phosphorylated E1-α subunit of BCKDC ascompared with the saline treated mice (p<0.05) (FIG. 5).

Example 5 Unaltered BCKDC Transcript Levels Following PBA Treatment

Since PBA is a known histone deacetylase (HDAC) inhibitor, transcriptlevels of BCKDC were measured following treatment with PBA usingquantitative RT-PCR. Five wild type mice were treated with eitherplacebo or PBA. Following treatment the liver and muscle cells wereharvested. RNA was isolated and subject to real time RT-PCR to assessthe transcript levels of all the subunits of the BCKDC and regulatorykinase. The transcript levels of the E1A, E1B, E2 were found to besignificantly reduced (p<0.006) in the liver. Hence, the observed PBAinduced increased enzymatic activity of BCKDC is not due to an increasein the amount of the enzyme (since there is a decrease in transcriptionfollowing PBA treatment), rather is due to the post-translationalmodification (decreased phosphorylation) of the existing protein. Nosignificant changes in the transcript levels of any of the subunits ofthe BCKDC or in the levels of the BDK (BCDKC kinase) were observedbetween the placebo and the PBA treated samples (FIG. 6). Hence, PBAinduced increased enzymatic activity of BCKDC is not due to the knowneffects of PBA as a histone deacetylase (HDAC) inhibitor.

Example 6 Phenylbutyrate Enhances E1 Activity and Inhibits E1αPhosphorylation and Inactivation.

To determine the effect of phenylbutyrate on individual BCAA catabolicenzymes, activities of mitochondrial branched chain amino transferase(BCATm) and BCKDC enzymes were measured with and without phenylbutyrateusing purified recombinant enzymes. BCATm generates the BCKA productsthat are elevated in MSUD and are the substrates for BCKDC. BCATmactivity was measured at pH 8.0 and 298 K as described in Example 2.Kinetics of the E1 decarboxylase reaction with and withoutphenylbutyrate were determined in the presence of an artificial electronacceptor 2, 6-dichlorophenolindophenol (DCPIP). The assay mixturecontained 100 mM potassium phosphate, pH 7.5, 2.0 mM MgCl₂, 0.2 mMthiamin diphosphate (ThDP) and 0.1 mM DCPIP. The rate of decarboxylationat 30° C. was measured by monitoring the reduction of the dye at 600 nm(24, 25). For the overall BCKDC activity assay, the enzymes wereexchanged into phosphate buffer (30 mM potassium phosphate, pH 7.5)containing 5 mM DTT using a PD-10 column and the enzyme concentrationswere calculated from the absorption maxima at 280 nm. The proteincomplex was reconstituted with E1, lipoylated E2 (lip-E2) and E3 at amolar ratio of 12:1:55, in which lip-E2 exists as a 24-mer. The assaymixture contained 30 mM potassium phosphate pH 7.5, 100 mM NaCl, 3 mMNAD⁺, 0.4 mM CoA, 2 mM MgCl₂, 2 mM DTT, 0.1% Triton X-100, and 2 mMThDP. The overall reaction was monitored by formation of NADH at 340 nm.The apparent rate constants (k_(app)) at different substrateconcentrations for all of the above assays were determined from theabsorption changes at the individual wavelength maximum. The k_(app)rate constants were fit using the following equation:

k _(app) =k _(cat)[S]/(K _(m)+[S])

The phosphorylation of E1 was carried out in the phosphorylationreaction mix (30 mM HEPES, pH 7.4, 2 mM DTT, 1.5 mM MgCl2, and 0.2 mMEGTA) with and without addition of phenylbutyrate. E1, E2, and E3proteins were mixed at 12:1:55 molar ratio in a 0.1 ml reaction mix, and0.1 μg of maltose-binding protein-tagged rat BCKDC kinase (BDK) wasadded. The mix was pre-incubated at room temperature for 15 minutes. Thephosphorylation reaction was started after addition of 0.4 mM ATP to thereaction mix, and the reaction was terminated at different time pointsby addition of higher salt concentration. Overall BCKDC activity wasmeasured as described above.

As shown in Table 5, there was no effect of phenylbutyrate on BCATmkinetics including k_(cat) and K_(m). BCKDC has multiple enzymeactivities and therefore, we tested the following enzyme activities inthe presence and absence of phenylbutyrate: E1 decarboxylase—bothunphosphorylated (fully active) and completely phosphorylated (inactive)E1, ability of BCKDC kinase to phosphorylate and inactivate E1, andoverall BCKDC activity. As shown in Table 6, phenylbutyrate enhancedunphosphorylated E1 (fully active enzyme) catalyzed decarboxylation ofall three BCKA significantly. It not only increased the k_(cat) but alsoincreased the sensitivity of the enzyme to BCKA by lowering their K_(m)values. Phenylbutyrate did not have an effect on phosphorylated E1(inactive enzyme), but did prevent completely phosphorylation andinactivation of E1 in the presence of BCKDC kinase (Table 5, comparek_(cat) and K_(m) values for phosphorylated E1 and E1 plusphenylbutyrate and BDK). Phenylbutyrate enhanced overall BCKDC activityas shown by the increases of 50% to 70% in k_(cat) values for BCKAs(Table 6). Therefore, phenylbutyrate appears to activate decarboxylaseactivity and increase the activity state of BCKDC by blocking kinasecatalyzed inactivation of E1.

TABLE 5 Phenylbutyrate protected E1 from BDK induced inactivation andhad no effect on the activity of phosphorylated E1^(a). E1 catalyzeddecarboxylase activity k_(cat) (min⁻¹) K_(m) (μM) Additions KIC KMV KIVKIC KMV KIV [−] Phenylbutyrate  7.6 ± 1.0  5.2 ± 0.8 12.0 ± 1.2 39.0 ±2.0 45.0 ± 4.0 48.0 ± 3.0 [+] Phenylbutyrate 20.2 ± 1.5 18.0 ± 1.0 25.0± 2.0 24.0 ± 2.0 21.0 ± 3.0 22.0 ± 2.0 [+] Phenylbutyrate^(b) 19.8 ± 1.720.0 ± 1.1 28.0 ± 1.9 27.0 ± 2.0 18.0 ± 2.0 20.0 ± 3.0 [−] BDK [+]Phehylbutyrate^(b) 20.6 ± 2.5 22.0 ± 1.8 26.0 ± 2.2 21.0 ± 1.9 21.0 ±3.0 25.0 ± 2.0 [+] BDK E1 catalyzed decarboxylase activity measuredafter inactivation by BDK^(b) [−] Phenylbutyrate  0.9 ± 0.1  0.6 ± 0.1 0.5 ± 0.1 532.0 ± 35.0 610.0 ± 28.0 680.0 ± 20.0 [+] Phenylbutyrate 0.9 ± 0.1  0.6 ± 0.1  0.6 ± 0.1 550.0 ± 27.0 642.0 ± 38.0 720.0 ± 47.0^(a)E1 protein was reconstituted with phenylbutyrate (1.0 mM) first andthen BCKD kinase (BDK) (0.1-0.5 μg) and ATP (0.4-1.0 mM) were added.^(b)E1 protein was phosphorylated first with the addition of BDK (0.1μg) and ATP (0.4 mM). Abbreviations: KMV, α-keto-β-methylvalerate; KIC,α-ketoisocaproate; KIV, α-ketoisovalerate.

TABLE 5 Phenylbutyrate enhanced overall BCKDC activity^(a). Activity ofBCKDC^(b) k_(cat) (min⁻¹) K_(m) (μM) Additions KIC KMV KIV KIC KMV KIV[−] Phenylbutyrate 140.0 ± 15.0 118.0 ± 10.0 197.0 ± 12.0 45.0 ± 6.053.0 ± 7.0 55.0 ± 4.0 [+] Phenylbutyrate 255.0 ± 10.0 226.0 ± 18.0 309.0± 15.0 41.0 ± 5.0 50.0 ± 3.0 40.0 ± 5.0 Abbreviations: KMV,α-keto-β-methylvalerate; KIC, α-ketoisocaproate; KIV, α-ketoisovalerate.

Thus, the present invention provides a novel mechanism of action of PBAfor the regulation of BCKDC enzymatic activity that affects thepost-translational modification of this protein complex, namelydephosphorylation, thereby increasing its enzymatic activity. Theincreased enzymatic activity of BCKDC thus obtained is effective intreatment of diseases related to the defective catabolism of branchedchain amino acids.

For example, the global action of phenylbutyrate and its derivatives onglobal phosphorylation may be applied to the treatment of diseases wherephosphorylation status of target proteins are regulated byphosphorylation wherein alteration of phosphorylation status of theprotein may lead to either decrease or increase in enzymatic activity.This alteration of enzymatic activity would translate intotherapeutically beneficial endpoints in the associated disease process.

Example 7

Enzyme Activity in Control and Patient Lymphoblast Cell Lines.

Following treatment with phenylbutyrate, enzyme activity in control andpatients' lymphoblast cell lines was measured. Transformed lymphoblastcell lines were available from all 5 MSUD patients and from 2 controlsto measure BCKDC activity. Lymphoblasts were incubated for 48 h with andwithout 1 mM phenylbutyrate (a lower concentration of phenylbutyrate wasused because of higher sensitivity of these cells as compared tofibroblasts to the drug) then BCKDC activity was measured withoutα-chloroisocaproate addition (CIC). As shown in FIG. 3, culturinglymphoblasts from controls with phenylbutyrate significantly enhancedleucine oxidation in both control (FIG. 7A) and patient lymphoblasts(FIG. 7B), with the exception of patient 5 in which the increase was notsignificant. Western blotting (FIG. 7C) with antibodies that detect E1α,E1α-P, E2, and the BCAT isozymes revealed a complex effect ofphenylbutyrate. Of the 3 patient responders, lymphoblasts from patients4 and 5 exhibited a decrease in the phosphorylation state of E1α with noapparent change in E1 levels. Neither the controls nor patients 1, 2 and3 responded. Phenylbutyrate also appeared to influence the levels of theBCAT isozymes, particularly the cytosolic isozyme BCATc which wasexpressed in lymphoblasts and appeared to increase in response tophenylbutyrate in all of the lymphoblasts but patient 5 lymphoblastcells. The levels of the mitochondrial isozyme, BCATm, were slightlyelevated in both controls as well as patients 1 through 3 uponphenylbutyrate treatment. Thus, in cells from the two patients with E2mutations (Table 7), activation of enzyme activity results frominhibition of phosphorylation state of E1α, in specific embodiments ofthe invention. In the other patient cell lines, enhanced activity didnot appear to correlate with phosphorylation changes but was ratherrelated with increased levels of the BCAT isozymes.

TABLE 7 Characteristics of the MSUD patients. Fibroblast BCKDCLymphoblast BCKDC activity^(a) activity^(a) DNA analysis % of % ofAffected Age normal normal enzyme (years) Gender Mean ± SD control Mean± SD control subunit Allele 1^(b) Allele 2^(b) Patient 1 24 Male   3 ±2.81 0.96 463.6 ± 32.1  9.13 E1α p.G290R p.G290R (p.G245R)^(c)(p.G245R)^(c) Patient 2 17 Male 7.4 ± 6.7 0.9 560.6 ± 98.8  11.04 E1αc.887_894del^(d) p.Y438N (p.Y393N)^(d) Patient 3 5 Female   2 ± 0.740.26 689.9 ± 72   13.59 E1α p.V412M p.V412M (p.V367M)^(e) (p.V367M)^(e)Patient 4 6 Female 272 ± 31  36.1 157.1 ± 100.7 3.09 E2 c.75_76del^(f)p.R301C (p.R240C)^(g) Patient 5 16 Female 7 ± 1 1.62 73.7 ± 18.1 1.45 E2p.S366P Exon 11 del^(g) (p.S305P)^(g) ^(a)Enzyme activity measured onfibroblasts or lymphoblasts is expressed in pmol CO₂ released/mgprotein/hour. ^(b)The numbering systems of amino acid residues beginningwith the initiation Methionine as +1 or with the amino terminus (inparenthesis) are both listed. ^(c)This mutation was previously reportedin homozygous state by Chuang et al. (1995) in patients with anintermediate form of MSUD. ^(d)Mutations previously reported by Zhang etal. (1989) and Chuang et al. (1995) in a patient with classic MSUD.^(e)Mutations previously reported by Henneke et al. (2003) in patientwith classic MSUD. ^(f)Mutation previously reported by Fisher et al.(1993) in compound heterozygous state with the p.E163X mutation in apatient with classic MSUD. ^(g)Mutations not previously reported.

Example 8

Phenylbutyrate Enhancement of BCAA Oxidation Involves Both Effects onPhosphorylation State of E1 and a Direct Action on Enzyme Activity

Alpha-chloroisocaproate (CIC) is a known inhibitor of branched-chainalpha-keto acid dehydrogenase kinase (BDK) (Harris et al., 1982) and isadded routinely when assaying BCKDC activity in human cell lines.Because phenylbutyrate affects E1α phosphorylation state in vivo (seeFIG. 5), leucine oxidation was measured in the same cell lines as inFIG. 8 with CIC in the assay. Both control cells exhibited higheractivity in the presence of CIC than alone and oxidation was increasedby phenylbutyrate (FIGS. 8A and 8B). In the patient samples, patients 1and 2 showed a similar pattern of response to phenylbutyrate as observedin the absence of CIC. On the other hand, in patient 3 lymphoblastleucine oxidation was higher than in the other cell lines and ratesnearly doubled in the cells incubated with phenylbutyrate. Westernblotting (FIG. 8C) with antibodies that detect E1α and E1α-P showed thatCIC alone, or together with phenylbutyrate, decreases thephosphorylation state of E1α in both controls and patients 4 and 5 buthas no effect on patients 1, 2, and 3. CIC did not have any effect on E2or the BCAT isozyme levels. Taken together, the results indicate thatthe ability of phenylbutyrate to enhance BCAA oxidation involved botheffects on phosphorylation state of E1 and a direct action on enzymeactivity. Changes in BCAT isoenzyme activity also impacts BCKAproduction, in specific cases. All patient cell lines accumulated moreKIC than observed in control cell lines.

REFERENCES

All patents and publications mentioned in this specification areindicative of the level of those skilled in the art to which theinvention pertains. All patents and publications herein are incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by referencein their entirety.

Patents and Patent Applications

-   U.S. Patent Application Publication US2010/0008859-   PCT Publication WO/2008/083226-   U.S. Pat. No. 6,613,308-   U.S. Pat. No. 5,641,515-   U.S. Pat. No. 5,580,579-   U.S. Pat. No. 5,792,451-   U.S. Pat. No. 5,466,468-   U.S. Pat. No. 5,543,158-   U.S. Pat. No. 5,641,515-   U.S. Pat. No. 5,399,363-   U.S. Pat. No. 5,629,001

PUBLICATIONS

-   Chuang, J. L., J. R. Davie, J. M. Chinsky, R. M. Wynn, R. P. Cox,    and D. T. Chuang. Molecular and biochemical basis of intermediate    maple syrup urine disease. Occurrence of homozygous G245R and F364C    mutations at the E1 alpha locus of Hispanic-Mexican patients. J Clin    Invest 95:954-63 (1995).-   Fisher, C. W., C. R. Fisher, J. L. Chuang, K. S. Lau, D. T. Chuang,    and R. P. Cox. Occurrence of a 2-bp (AT) deletion allele and a    nonsense (G-to-T) mutant allele at the E2 (DBT) locus of six    patients with maple syrup urine disease: multiple-exon skipping as a    secondary effect of the mutations. Am J Hum Genet 52:414-24 (1993).-   Harris, R. A., R. Paxton, and A. A. DePaoli-Roach. Inhibition of    branched chain alpha-ketoacid dehydrogenase kinase activity by    alpha-chloroisocaproate. J Biol Chem 257:13915-8 (1982).-   Henneke, M., N. Flaschker, C. Helbling, M. Muller, P.    Schadewaldt, J. Gartner, and U. Wendel. Identification of twelve    novel mutations in patients with classic and variant forms of maple    syrup urine disease. Hum Mutat 22:417 (2003).-   Recommended Dietary Allowances”, 10th ed., Food and Nutrition Board,    National Academy of Sciences, 1989-   Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,    1990.-   Zhang, B., H. J. Edenberg, D. W. Crabb, and R. A. Harris. Evidence    for both a regulatory mutation and a structural mutation in a family    with maple syrup urine disease. J Clin Invest 83:1425-9 (1989).

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the invention asdefined by the appended claims. Moreover, the scope of the presentapplication is not intended to be limited to the particular embodimentsof the process, machine, manufacture, composition of matter, means,methods and steps described in the specification. As one will readilyappreciate from the disclosure, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized. Accordingly, the appended claims areintended to include within their scope such processes, machines,manufacture, compositions of matter, means, methods, or steps.

What is claimed is:
 1. A method of treating an individual having ametabolic disorder from an inborn error in metabolism of one or morebranched chain amino acids, comprising administering to the individual atherapeutically effective amount of a composition comprising one or moreammonia scavengers.
 2. The method of claim 1, wherein the metabolicdisorder is selected from the group consisting of maple syrup urinedisease (MSUD), hypervalinemia, isobutyryl-CoA dehydrogenasedeficiency,⋅beta-ketothiolase deficiency, 2-Methylbutyryl-CoAdehydrogenase deficiency, hypermethioninemia, homocystinuria,cystathioninuria, isovaleric acidemia, 3-Methylcrotonyl-CoA carboxylasedeficiency, or 3-hydroxy-3-methylglutaryl-CoA lyase deficiency, FragileX, tuberous sclerosis, Rett syndrome, autism, diabetes, and acombination thereof.
 3. The method of claim 1, wherein the metabolicdisorder comprises an increase in blood plasma levels of branched chainamino acids and/or branched chain alpha-keto acids.
 4. The method ofclaim 3, wherein the branched chain amino acid is at least one ofleucine, isoleucine, and valine.
 5. The method of claim 3, wherein thebranched chain alpha-keto acid is at least one of keto-isocaproic acid,keto-methylvaleric acid, and ketoisovaleric acid.
 6. The method of claim1, wherein the ammonia scavenger is selected from the group consistingof phenylbutyrate, BUPHENYL® (sodium phenylbutyrate), AMMONAPS®,butyroyloxymethyl-4-phenylbutyrate, glyceryl tri-[4-phenylbutyrate](HPN-100), esters, ethers and a combination thereof,
 7. The method ofclaim 1, wherein the ammonia scavenger is the native form, a salt, anacid, and/or a prodrug of a native ammonia scavenger.
 8. The method ofclaim 7, wherein the salt is the sodium salt, calcium salt, lithiumsalt, potassium salt, or a mixture thereof.
 9. The method of claim 1,wherein the ammonia scavenger is at least one compound of the formula:

wherein n 0, 2, 4, 6 or 8, or a pharmaceutically acceptable salt orester or prodrug thereof.
 10. The method of claim 1, wherein theadministration of the ammonia scavenger results in a decrease in bloodplasma levels of branched chain amino acids and/or branched chainalpha-keto acids.
 11. The method of claim 1, wherein administration ofthe ammonia scavenger stimulates the baseline enzymatic activity of thebranched chain dehydrogenase enzyme complex protein to levels effectivein achieving decreased branched chain amino acid and/or branched chainalpha-keto acid levels.
 12. The method of claim 10, wherein thestimulation of activity is caused by a decrease in the phosphorylationof S293 and S303 residues of the E1□□ subunit of the branched chaindehydrogenase enzyme complex.
 13. The method of claim 1, furthercomprising assaying for a decrease in plasma levels of at least one ofsaid branched chain amino acids and/or assaying for a decrease in plasmalevels of branched chain alpha-keto acids.
 14. A method for stimulatingthe baseline enzymatic activity of the branched chain dehydrogenaseenzyme complex protein in an individual comprising administrating atherapeutically effective amount of one or more ammonia scavengers tothe individual.
 15. The method of claim 14, wherein the stimulation ofactivity is caused by a decrease in the phosphorylation of S293 and S303residues of the E1□□ subunit of the branched chain dehydrogenase enzymecomplex.
 16. The method of claim 14, wherein the individual has aninborn error of metabolism.
 17. The method of claim 14, wherein theindividual has an accumulation of branched chain amino acids and/orbranched chain alpha-keto acids in the individual's blood plasma. 18.The method of claim 14, wherein the ammonia scavenger is a compositionselected from this list consisting of phenylbutyrate, BUPHENYL® (sodiumphenylbutyrate), AMMONAPS®, butyroyloxymethyl-4-phenylbutyrate, glyceryltri-[4-phenylbutyrate] (HPN-100), esters, ethers and a combinationthereof, wherein the composition is the native form, a salt, an acid,and/or a prodrug of the native composition.
 19. The method of claim 18,wherein the salt is the sodium salt, calcium salt, lithium salt, and/orpotassium salt.
 20. The method of claim 14, wherein the ammoniascavenger is at least one compound of the formula:

wherein n 0, 2, 4, 6 or 8, or a pharmaceutically acceptable salt orester or prodrug thereof.
 21. The method of claim 14, wherein theadministration of the one or more ammonia scavengers comprises adecrease in blood plasma levels of branched chain amino acids and/orbranched chain alpha-keto acids.